SUMMARY Head and neck squamous cell carcinomas (HNSCCs) are the sixth most common cancer worldwide. Despite decades of research, afflicted patients continue to suffer a staggering ~50% five-year mortality rate. Two enormous obstacles impede the development of more effective therapies for HNSCC: 1) poor understanding of the pathways that lead to malignancy and 2) inability to detect tumors early enough. While much has been learned about the mutational landscape of HNSCCs through next generation sequencing, a tremendous challenge remains in translating this genomic information into functional relevance. Reliance on animal models of cancer is critical for separating driver from passenger mutations, to determine pathways that synergize to promote aggressive tumors, and to develop diagnostic/prognostic tools and therapeutic targets. Unfortunately, the ability to develop and rapidly test these models is hampered by the large investment of time and resources required to generate transgenic mice. This proposal addresses these significant knowledge gaps through the synergistic implementation of cutting-edge genetic and imaging techniques developed by the two principal investigators. Our long-term goal is to establish high-throughput mouse models of oral cancer that will elucidate cryptic pathways that promote tumor growth, providing insights into new avenues for therapy. Here, we adopt our versatile in utero lentiviral transduction technique and novel LumiFluor bioluminescence resonance energy transfer (BRET) reporter to a validated model of oral cancel, to manipulate underexplored genetic pathways revealed by genome-wide HNSCC sequencing studies, and visualize how they affect tumor growth kinetics. Our objective is to understand how self-renewal and differentiation pathways critical to normal development are co-opted by cancer cells. Our rationale is that two-thirds of HNSCC patient tumors show mutations in differentiation genes, which is correlated with poor patient survival. Based on our compelling preliminary studies, we will test the central hypothesis that imbalance between symmetric and asymmetric cell divisions in tumors?mediated by cytoskeletal control of the spindle orientation machinery and epigenetic regulation of ?stem-ness??play important roles in HNSCC tumorigenesis. Our Specific Aims are to: 1) Determine the function of spindle orientation genes in oral carcinogenesis, as this pathway directly regulates self-renewal/differentiation decisions; and 2) Establish a high-throughput in vivo functional assay for modulators of tumorigenesis. Both aims use our improved Cre-inducible mouse model of oral cancer based on a validated, established tumorigenesis protocol driven by high-risk HPV16 E6/E7 and the chemical carcinogen 4-NQO. The proposed research plan is both technically and conceptually innovative. The idea that spindle orientation is an important regulator of solid tumor growth kinetics is untested, and we possess a unique toolkit?in utero lentiviral RNAi for rapid genetic manipulations, LumiFluor reporter for advanced imaging, and an inducible model of HPV+ HNSCCs?that will allow us to make important insights into HNSCC biology.